Diagnostic methods involving determining gene copy numbers and SNPs in the FcyRII/FcyRIII gene cluster, and probes for use in such methods to detect susceptibility to and treatment efficacy in autoimmune diseases

ABSTRACT

The invention relates to diagnostic methods to predict whether a subject is predisposed for acquiring a disease or to predict the therapy responsiveness of an individual patient. Provided is a method for determining whether a subject is predisposed for developing an autoimmune disease, comprising determining in a sample isolated from said subject the amount of intact genes, or gene products thereof, of the FcγRII/FcγRIII gene cluster, said gene cluster comprising the FCGR2C, FCGR3A, FCGR2A and FCGR3B genes encoding an activating FcγR, and FCGR2B encoding an inhibitory Fcγ R; and correlating said amount to the amount observed in a healthy population. Also provided is a method to predict the responsiveness of a subject to therapy with intravenous immunoglobulin (IVIg) therapy or a monospecific biological, such as a humanized or human monoclonal antibody or a chimeric molecule, comprising the C-terminal Fc-tail of IgG.

This application is the U.S. National Phase of, and Applicants claimpriority from, International Application Number PCT/NL2006/000662 filed22 Dec. 2006, and European Application bearing Serial No. 05077984.2filed 22 Dec. 2005, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to the fields of immunology and molecularmedicine. In particular, it relates to diagnostic methods involving thedetermination the amount of intact genes of the FcγRII/FcγRIII genecluster and to uses thereof in disease management, for example topredict whether a subject is predisposed for acquiring a disease or topredict the therapy responsiveness of an individual patient. It alsorelates to probes and nucleic acid constructs for use in such methods.

Sequencing the human genome has prompted many technical advances andstimulated the discoveries of various genomic landmarks. One area is thedevelopment of a dense set of polymorphic markers for gene mapping. Themost common form of DNA sequence variation is a single nucleotidepolymorphism (SNP) but, in addition, there are microsatellite repeatpolymorphisms and insertion/deletion polymorphisms. Whereas SNPs andmicrosatellites have been very well characterized in terms of theirgenomic locations and their frequencies in different populations,insertion/deletion polymorphisms are less well characterized. Recentpapers (see for example Sebat J. et al. Large-scale copy numberpolymorphism (CNP) in the human genome. Science 2004; 305: 525-528)described the identification of large (several kilobases to megabases)deletions and duplications of DNA fragments when genomes of normalindividuals were compared. The identification and characterization ofCNPs show that in addition to single-nucleotide differences, genomes ofunrelated individuals have large regions of thousands to millions ofnucleotides that are different.

From a functional perspective, gene copy number differences cancontribute to variation in gene expression. CNPs of coding andregulatory regions are likely to affect expression of genes at thetranscript and/or protein levels. Gene expression studies have shownthat subtle differences in expression levels of genes have significantconsequences. For example, copy number polymorphism of the defensin genecluster probably accounts for natural variation in the expression levelof DEFB4 which, in turn, might account for individual differences inimmune defence (Linzmeier et al., Genomics. 2005; 86:423-30).Furthermore, Gonzalez et al. (Science. 2005; 307:1434-40) reported thatpossession of a CCL3L1 copy number lower than the population average isassociated with markedly enhanced HIV/acquired immunodeficiency syndrome(AIDS) susceptibility. Cappuzzo et al. (J. Natl Cancer Inst. 2005;97:643-55) disclosed that patients with advanced non-small-cell lungcancer whose tumour cells contain extra copies of the epidermal growthfactor receptor (EGFR) gene may be more likely to respond to the druggefitinib (Iressa), and suggested that this high gene copy number may bean effective predictor of gefitinib efficacy.

Thus, evidence is accumulating that CNPs and their overall effect ongene expression contributes significantly to the variation thatunderlies individual differences in predisposition to complex diseases.

SUMMARY OF THE INVENTION

It is a goal of the present invention to provide new diagnostic markersfor autoimmune diseases, in particular markers that can be used topredict whether a) a subject is predisposed for developing an autoimmunedisease and, b) once a subject has been diagnosed with an autoimmunedisease, whether the individual subject will follow a severe or a benigncourse of the disease or whether the disease will develop into a chronicform and, c) whether the individual subject diagnosed with an autoimmunedisease will respond to certain types of therapy.

These goals are met by the finding that genes of the FcγRII/FcγRIII genecluster are suitably used as prognostic markers for major autoimmunediseases, as well as for predicting disease development and/or therapyresponsiveness to intravenous immunoglobulin (IVIg) therapy. As isdescribed in detail below, the inventors set out to measure the copynumber and SNPs of genes in the human FcγRII and FcγRIII gene cluster atchromosome 1q21-23. Using primers designed to hybridize in the regionsof the FcγRII and RIII genes that are specific for the genes, the copynumber of FcγRII and RIII genes in human subjects suffering from anauto-inflammatory disease was determined. More specifically, dataobtained from patients suffering from an auto-inflammatory disease inchildhood—the so-called Kawasaki Disease- and from patients havingimmune thrombocytopenic purpura (ITP) were compared with those ofcontrol subjects.

Kawasaki Disease is an acute febrile syndrome in infancy, which ischaracterized by a vasculitis of mainly the medium-sized arteries. Sincethere is no specific test to diagnose the disease, diagnosis is made onclinical criteria. In this form of pediatric vasculitis, high-doses ofIVIg infusions are used as standard treatment to prevent coronary arterylesion. As is disclosed herein, Kawasaki patients were on average foundto have an amount different from the usual number of two FCGR2B genesper genome, when normalized to several reference genes (CYBB as X-linkedgene, SRY as Y-linked gene and ALB encoding the plasma protein humanserum albumin, as an autosomal gene). Variations of the gene copy numberin patients and control subjects are shown in the Examples below.

ITP is a disease characterised by thrombocytopenia with otherwise normalcell lineages and no other explanation for the isolatedthrombocytopenia. Destruction of autoantibody-sensitised platelets byFcγR-bearing phagocytic cells in the reticuloendothelial system plays animportant role, although the exact pathophysiology of this autoimmunedisorder is not precisely known. The role of Fc receptors is underscoredby the fact that intravenous immunoglobulin (IVIg) treatment, viablockade of the Fc receptors for IgG, and splenectomy (removal of theplatelet-destructing organ) are effective treatment options.

Previous studies concerning polymorphisms in the FCGR gene cluster inITP patients show conflicting results. In a study involving 116 ITPpatients, the present inventors found an increased variation in theFCGR2 and FCGR3 gene cluster in Caucasian patients as compared tohealthy Caucasian controls. For example, 82% of a healthy population ishomozygous for a SNP that converts a glutamine in the FGCR2C openreading frame (ORF) into a stop codon, rendering FCGR2C a non-expressedpseudogene. The remainder (18%) of the healthy population containsFCGR2-ORF. In contrast, 35% of the ITP patients were found to carry atleast one FCGR2C-ORF allele, which results in the expression of afunctionally activating FcγRIIc. Furthermore, a significant differencein genotype and allele frequency for the promoter polymorphism −386G/Cwas observed. The genotype −386CC was rare, and only observed in an ITPpatient. In the same series of DNA samples, we confirmed the previouslyobserved overrepresentation of the SNP in FCGR3A encoding theFcγRIIIa-158V variant, being most prevalent in ITP of childhood-onset(p=0.0005) and not ITP of adult-onset (p=0.3).

These studies indicate that the relative amounts of activating versusinhibitory FcγRs are indicative for developing an autoimmune disease. Ithas been previously reported that antibodies against FcγRII used couldmake a distinction between health and disease in autoimmunity orotherwise, but this is incorrect. Although the relevance of suchimbalance between activating and inhibitory FcγRII receptors has beenrepeatedly suggested, to date all these studies were based on the use ofnon-specific antibodies that, first of all, can not differentiatebetween the different FcγRII isoforms [Nakatani et al. Clin. Exp.Immunol. 1999; 117:418-22; Van Wijngaarden et al., Rheumatology 2003;42:681-8]. Moreover, the authors often refer to staining patterns bymonoclonal antibodies (MoAbs) on immune cells that are known to havehighly variable expression levels of FcγRs because of in-vivo orin-vitro activation by growth factors, cytokines or inflammatorytriggers. Furthermore, the present inventors show that the recentlydeveloped “FcγRIIb-specific MoAbs” that have always been assumed to bindexclusively to FcγRIIb, also bind to FcγRIIc-expressing cells. Becauseof the identical extracellular domains of FcγRIIb and FcγRIIc, theseobservations are to be expected in individuals carrying the uncommonFCGR2C-ORF allele and hence expressing FcγRIIc. In sum, a geneticapproach to quantify the variability in CNPs and SNPs in the FCGR genecluster, preferably in a single assay as proposed by the presentinventors, is the most reliable way of testing disease susceptibility,disease severity and treatment efficacy in IgG-mediated processes fromchanges in this gene cluster.

DESCRIPTION OF THE INVENTION

Herewith, the invention provides a method for determining whether asubject is predisposed for developing an autoimmune disease, comprisingdetermining in a sample isolated from said subject the amount of thegenes, or of gene products thereof, in the FcγRII/FcγRIII gene cluster,said gene cluster comprising the FCGR2C, FCGR3A, FCGR2A and FCGR3Bgenes, encoding an activating FcγR, and FCGR2B encoding an inhibitoryFcγR; and correlating said amount to the amount observed in a healthypopulation, wherein an increased amount of a gene encoding an activatingFcγR (i.e. FCGR2C, FCGR3A, FCGR2A and FCGR3B) or gene product thereof,and/or a decreased amount of gene encoding an inhibitory FcγR (i.e.FCGR2B), or gene product thereof, is indicative of having an increasedchance of developing an autoimmune disease.

The term “gene product” as used herein refers to products resulting fromexpression of said genes (RNA or protein) and thus comprises transcripts(mRNA) and translated proteinaceous substances encoded by one of theFCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B genes (also referred to asFcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb, respectively).Preferably, it refers to mRNA, in view of lack of specific probes whichcan distinguish between the gene products at the protein level.

The expression “activating FcγR” refers to an FcγR which is functionallyactive and capable of providing stimulatory or positive signalling. Italso comprises “hyperactive” variants or mutant receptors that display ahigher activity as compared to their normal counterpart, for exampleresulting from a SNP. Likewise, an “inhibitory FcγR” refers to an FcγRwhich is functionally active and capable of providing inhibitory ornegative signalling.

According to the invention, a gene can be a genomic sequence such as acDNA sequence. A “sample isolated from said subject” can be any type ofbiological sample or biopsy comprising cellular material, plasma, serum,urine, sputum, liquor, etc. of the subject. Nucleic acids can beextracted by methods known in the art to yield a nucleic acid sample,for example using commercial DNA or RNA extraction kits.

As used herein, the term “autoimmune disease” refers to any disease ordisorder in which the progression from benign autoimmunity to pathogenicautoimmunity occurs. An estimated 5-10% of all people suffer frominflammations or a reactivity that may in many instances be related tothe formation of antibodies against an individual's own tissuecomponents. Autoimmunity is caused by a complex interaction of multiplegene products, unlike immunodeficiency diseases, where a single dominantgenetic trait is often the main disease determinant. In one embodiment,the autoimmune disease is an organ-specific autoimmune disease.

Examples of auto-immune diseases are organ-specific (i.e. type 1diabetes mellitus (IDDM1), Addison's disease, Graves' disease,Hashimoto's thyroiditis, autoimmune hypoparathyroidism, autoimmunehypophysitis, autoimmune oophoritis, autoimmune orchitis, autoimmunepolyendocrinopathy, autoimmune syndrome type 1 (PAS-1), type 2 (PAS-2)and type 3 (PAS 3), polymyositis/dermatomyositis, bullous pemphigoid,epidermolysis bullosa acquisita, pemphigus foliaceous, pemphigusvulgaris, alopecia greata, vitiligo, autoimmune hearing loss, Meniere'ssyndrome, Mooren's ulcer, multiple sclerosis (MS), myasthenia gravis,Vogt-Koyanagi-Harada disease, autoimmune myocarditis, pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, celiac disease,inflammatory bowel disease (IBD), Crohn's disease, Guillain-Barrésyndrome, chronic inflammatory demyelinating polyneuropathy, IgAnephropathy, Goodpasture's syndrome,) In another embodiment, theautoimmune disease is of a systemic and/or hematological nature (i.e.idiopathic thrombocytopenic purpura, aplastic anemia, autoimmunehemolytic anemias, antiphospholipid syndrome, autoimmunelymphoproliferative syndrome, polyarteritis nodosa, polyglandularBechet's disease, Takayasu's arteritis, Kawasaki's disease (KD),rheumatoid arthritis (RA), sarcoidosis, scleroderma, Sjögren's syndrome,systemic lupus erythematosus (SLE), Reiter's syndrome, ankylosingspondilitis, Wegener's granulomatosis, Churg-Strauss syndrome, giantcells arteritis). In addition to these more or less ‘classical’ examplesof auto-immune disease, recent data indicate that some psychiatric oraffective disorders also have an inflammatory component.

In a preferred aspect, the invention provides a method for predictingthe predisposition to Kawasaki disease, Systemic Lupus Erythematosus(SLE), Idiopathic Thrombocytopenic Purpura (ITP) and/or rheumatoidarthritis (RA).

It is known that the intact FCGR2A, FCGR2C (e.g. FCGR2C-ORF), FCGR3A andFCGR3B genes encode activating FcγRII and FcγRIII receptors, whileFCGR2B encodes an inhibitory FcγRIIb. Without wishing to be bound bytheory, the inventors propose that a change in the balance between theamount or activity of activating FcγRII and FcγRIII receptors on the onehand and the inhibitory FcγRIIb receptor on the other hand is associatedwith autoimmune disease. This is in line with the identification ofpatients with auto-inflammatory Kawasaki disease (KD) or ITP havingthree instead of the usual two alleles of the FCGR3A gene for theactivating FcγRIIIa receptor, and the increase in FCGR2C-ORF observed inboth KD and ITP.

In a preferred embodiment, a method of the invention therefore comprisesdetermining the relative amount of activating versus inhibitory Fcγreceptors, and comparing the relative amount found in a matching healthycontrol population. An increase in the ratio of activating overinhibitory receptors is an indicator of having or developing anauto-immune disease.

It will be understood by the skilled person that a method of theinvention can involve the detection of different activating and/orinhibitory. genes or products thereof. In one embodiment, a method ofthe invention comprises determining the amount of intact FCGR2C gene orgene product, preferably FCGR2C-ORF. Since the FCGR2C is not expressedas a functional activating receptor in the majority of a healthypopulation being homozygous for the pseudogene of FCGR2C due to theFCGR2C^(stop) allele, the presence of an intact FCGR2C-ORF allele willshift the balance to a relative excess of stimulatory signals.

As indicated above, in a method according to the invention the amount ofan activating and/or inhibitory FcγRII or FcγRIII gene (product) can bedetermined at the genomic, transcriptional and/or the translationallevel (i.e. protein expression). Detection at the genomic level ispreferred since genomic analyses are typically fast, reliable and do notrequire highly specialized personnel. For example, it may entail thedetermination of the FcγR gene copy number polymorphisms (CNPs) and/orthe analysis of genomic alterations, for example SNPs, which directly orindirectly affect gene transcription or function of the gene product.

Methods for detecting CNPs are known in the art. The majority of theknown methods are based on quantitative polymerase chain reaction(Q-PCR) methods. The Q-PCR reaction is characterised by an exponentialincrease in the amount of PCR product in the early cycles, which reachesa plateau phase as the reaction products become inhibitory. The higherthe starting copy number of the target gene, the sooner the increase influorescence is detected and a lower threshold cycle number (Ct) valueis reached. The possibility of direct measurement of the PCR productaccumulated after each amplification cycle, without any intermediatesteps, ensures the high specificity of Q-PCR assays. Currently fourdifferent types of probes, TaqMan® (Applied Biosystems, Foster City,Calif., USA), Molecular Beacons, Scorpions®, FRET probes and theDNA-binding dye SYBR® Green (Molecular Probes), are generally used forQ-PCR. SYBR Green is a fluorogenic dye that exhibits little fluorescencewhen in solution, but emits a strong fluorescent signal upon binding todouble-stranded DNA.

Studies using Q-PCR for detecting gene copy number involve relativequantitative methods that rely on detection of a reference gene tocalculate the gene copy number, i.e. the gene of interest is normalizedto an appropriate endogenous (housekeeping) gene. To date, the ALB geneencoding albumin is generally being used for direct comparison andquantification as a golden standard reference. For example, Thiel et al.(2002) used ALB as an internal reference gene to detect the number ofgenes encoding peripheral myelin protein 22 (PMP22) on chromosome17p11.2-12, which gene is responsible for the hereditary neuropathyCharcot-Marie-Tooth (CMT1A). The same approach with albumin as internalstandard was applied by Schaeffeler et al. (2003) to determine theCYP2D6 gene dosage per genome to study drug metabolism phenotypes. BothThiel and Schaeffeler used Taqman technology with Taqman probes. Asimilar approach with ALB as a reference gene was undertaken by Layfieldet al. (2003) using LightCycler technology with SYBR Green to quantitatethe copy number of EGF receptor genes in colorectal adenocarcinoma.

In all these studies, the number of genes for albumin was assumed to be2 in total—i.e. one located on the maternal chromosome number 4 and oneon the paternal chromosome number 4 in any individual. This assumptionhas been accepted as a “fact” without proper verification or unequivocalproof. However, the present inventors observed that the gene copy numberof ALB varies considerably (2 or more) among human individuals and isthus not as stable as expected. It appeared that the ALB gene could notbe used as reliable reference gene in gene dosage studies. Therefore,they set out to identify a reference gene that has a lower variance incopy number as compared to the “golden standard ALB gene” encoding humanserum albumin, which can serve as a more reliable reference gene in genedosage studies. This goal was met by the observation that the CYBB gene,localized in humans on the X chromosome, has a remarkable low variancein copy number. Using CYBB-specific primers that anneal in or aroundexon 8 of the CYBB gene, the inventors screened more than 100 controls(males and females), more than 200 obligate carriers (females), morethan 300 male patients, and about 30 foetuses as a routine prenataldiagnostic effort. This screening revealed no variance in CYBB copynumber at all.

Accordingly, the invention provides a method for determining the copynumber of the FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B target genes, ororthologues thereof, in the genome of a subject, comprising detectingthe amount of said target genes relative to the amount of a referencegene in a nucleic acid sample of said subject, wherein the referencegene is the CYBB gene or an orthologue thereof. Preferably, the subjectis a mammalian subject, more preferably a human subject.

The CYBB gene (gene aliases are CGD; NOX2; GP91-1; GP91PHOX wasidentified at Xp21. It encodes the CYBB protein, also known asglycoprotein-91 (gp91)-phox, cytochrome b₅₅₈, beta polypeptide,cytochrome b.₂₄₅, beta polypeptide. Cytochrome b₅₅₈ is composed ofcytochrome b₅₅₈ alpha (CYBA) and beta (CYBB) chain. It has been proposedas a primary component of the microbicidal oxidase system of phagocytes.CYBB deficiency is one of six described biochemical defects associatedwith chronic granulomatous disease (CGD).

The method of the invention preferably involves the detection of one ormore target genes and said CYBB gene using Q-PCR technology.

Other methods for quantitative nucleic acid analysis can also be used,such as Southern blotting or multiplex ligation-dependent probeamplification (MLPA). The skilled person will be able to design suitablesets of PCR primers for the detection of the target gene(s) and the CYBBgene. In one embodiment, CYBB is detected using CYBB-specific primers inor around exon 8, as this was found to give a high PCR efficiency.Suitable probes for detecting amplified PCR products include SYBR GreenI Dye, hydrolysis probes or hybridisation probes. The two most usedquantitative PCR technologies are the Taqman™ technology of AppliedBiosystems and the LightCycler® technology of Roche. In one aspect,LightCycler®™ technology is employed. The system works with glasscapillaries. Hot and cold air allowing rapid ramping—30 cycles can beperformed within 20 minutes. The PCR process is monitored byfluorescence quantification of DNA-binding dyes for general detection ofdouble-stranded DNA, or, with hybridization probes, to monitor theamount of a specific target sequence. The probes are irradiated with ablue-light-emitting diode (470 nm), which excites yellow dyes includingfluorescein (FAM, FITC) and SybrGreen. The emitted fluorescent light isdetected in three channels for green light (530 nm, fluorescein), redlight (640 nm, LightCycler® Red 640) and near infrared (710 nm, LC Red705).

In a preferred embodiment, a method of the invention comprisesdetermining the copy number of one or more intact target genes of theFcRII/FcγRIII gene cluster by PCR, using a recombinant nucleic acidconstruct (“reference construct”) comprising a known number of copies ofgenes selected from the group consisting of FCGR2A, FCGR2B, FCGR2C,FCGR3A and FCGR3B, or orthologues thereof, fragment(s) thereof as wellas a known number of copies of the reference gene, or fragment thereof.Therefore, the invention provides a recombinant nucleic acid “referenceconstruct” comprising two segments, a first segment comprising the geneof interest selected from the group consisting of FCGR2A, FCGR2B,FCGR2C, FCGR3A and FCGR3B, or orthologues thereof, or a fragmentthereof, and a second segment comprising a reference gene, or a fragmentthereof, preferably wherein the ratio of said first to said secondfragment is 1:1.

The reference construct is for example a plasmid or vector comprisingsaid at least said first and second segment. The first segment with thetarget gene sequence(s) and the second segment with the reference genesequence can be ligated substantially directly to each other or they canbe spaced by a stretch of amino acids. The spacing between the twosegments is not of particular relevance as long as both segments arepresent on the same construct. The order of the first and second segmentwithin the construct is irrelevant. For reasons given above, a nucleicacid construct of the invention preferably comprises as a reference genethe human CYBB, or a relevant orthologue thereof in case the copy numberof a non-human subject is to be determined. Orthologues of human CYBBare known in the art. In a specific aspect, the reference constructcomprises at least 50 base pairs of human CYBB. It was found thatligation of 198 bp of exon 8 of the CYBB gene to a fragment of a targetgene (e.g. FCGR gene, see further below) gave very good results.

From the comparison of the reference gene (segment) and the target gene(segment) in a known ratio (e.g. 1:1) on the one hand with the targetDNA containing an unknown gene copy number for these genes, the exactdifferences in PCR amplification efficiency and therefore the exact generatio's can be calculated from the LightCycler or Taqman technique. Amajor advantage of using a reference construct of the invention whendetermining the copy number of a target gene resides in the fact thatthe external construct containing both target and reference gene inevery run corrects for differences in PCR amplification efficiency andgene quantity caused by variations in the initial sample amount. Thisapplication makes the gene dosage analysis robust and trustworthy.

Therefore, in one embodiment the invention provides a method fordetermining whether a person is predisposed for developing an autoimmunedisease as described herein above, said method comprising determining,preferably by Q-PCR, the amount of FCGR2C, FCGR2B, FCGR2A, FCGR3A andFCGR3B target genes (or orthologues thereof in case the method isapplied to non-human subjects), and the presence of a reference gene ina nucleic acid sample of said subject, using as external control arecombinant nucleic acid construct comprising at least a fragment of thetarget gene and a fragment of the reference gene.

The skilled person will understand that a detection method of theinvention using CYBB as reference gene and/or the recombinant controlDNA is not limited with respect to a particular target gene or group oftarget genes of which the copy number is to be determined.

In one embodiment, a method of the invention involves SNP analysis ofone or more regions, e.g. the promoter region, controlling the rate andextent of FcγR gene transcription. Polymorphisms in the promoter regionat nucleotide−386 and −120 are linked to transcriptional activity of theFCGR2B gene (Su et al., J Immunol. 2004; 172:7186-91). Four differenthaplotypes have been identified, i.e. 2B.1 or “wild type” (−386G/−120T);2B.2 (−386C/−120T), which may be a more frequent polymorphism in thepromoter for the FCGR2C (pseudo)gene; 2B.3 (−386G/−120A); and 2B.4(−386C/−120A). The 2B.4 haplotype was reported to have enhancedtranscriptional activity compared to the 2B.1 haplotype. The differencesin the ratios of FcγRIIa/FcγRIIb2 mRNA in neutrophils (see furtherbelow) were found to be related to these polymorphisms. A strongassociation with the 2B.4 haplotype and the 1:1 FcγRIIa/FcγRIIb2 mRNAratio was found, whereas the 2:1 ratio was highly associated and the 3:1and 4:1 FcγRIIa/FcγRIIb2 mRNA ratios even strictly associated with the2B.1 haplotype with low transcriptional activity. These findingsindicate that the increased transcription of FCGR2B in neutrophils isdependent to the 2B.4 haplotype, resulting in lower FcγRIIa/FcγRIIb2mRNA ratios. The fact that mononuclear cells of the same donors showwidely variable and non-fixed FcγRIIa/FcγRIIb2 ratios, demonstratesadditional genetic factors to be identified.

A PCR method of particular interest for the present invention involvesmultiplex ligation-dependent probe amplification (MLPA) technology,originally described by Schouten et al. (Nucleic Acids Res 2002;30:e57). MPLA is highly specific and requires a minimum of 20 ng ofgenomic DNA to be analysed. For instance, it can be used for therelative quantification of 40 different DNA sequences in an easy toperform reaction and uses more than one reference gene. Knownapplications of MLPA technique include the detection of exon deletionsand duplications in the human oncogenes, detection of trisomies such asDown's syndrome, characterisation of chromosomal aberrations in celllines and tumour samples as well as SNP or mutation detection. Relativequantification of mRNAs by MLPA is another application describedelsewhere (Eldering et al., Nucleic Acids Res 2003; 31:e153).

In MLPA, not the sample nucleic acids but the probes that are added tothe samples are amplified by PCR and quantified. Amplification of theprobes depends on the presence of probe target sequences in the sample.Each probe set consists of two oligonucleotides, two synthetic probes orone synthetic probe and one M13-derived probe that hybridize to adjacentsites of the target sequence. These hybridized probes are ligated,permitting subsequent amplification. All ligated probes have identicalend sequences that allow simultaneous PCR amplification by the use ofone universal primer pair. By varying the probe length for each probe,this will give rise to an amplification product of unique size between130 and 480 bp. Probe target sequences are typically small (50-100nucleotides). The prerequisite of a ligation reaction provides theopportunity to discriminate between single nucleotide differences. SNPanalysis is conveniently done by MLPA technology using target-specificMLPA probes. The inventors designed two novel mixes of MLPA probes todetect FCGR-specific sequences within exons, introns and promoterregions, as well as to detect FCGR2B-, FCGR2C-, FCGR3A-, andFCGR3B-specific SNPs (see FIG. 1 and Table 2). This is a majorachievement, since the FCGR2A, FCGR2B and FCGR2C genes show an extremelyhigh degree of homology, as do the FCGR3A and FCGR3B genes. In oneembodiment, the invention provides a method for the CNP and/or SNPdetection at the genomic level by MLPA analysis, wherein at least one ofthe probes as indicated in bold in Table 2A or 2B is used. Preferably,it comprises the use of all probes indicated in bold in Table 2A and/or2B. More preferably, it comprises, in one reaction tube, the use of allprobes of Table 2A (Probe Mix 1) and in a separate reaction tube theprobes of Table 2B (Probe Mix 2). Tables 2C, 2D and 2E show the targetsequences that are detected by the probes of Tables 2A and 2B, i.e. theprobe sequence is complementary to the target sequence. Also providedherein is an MLPA probe that is suitably used in a method of theinvention, said probe being selected from Tables 2C, 2D or 2E. In aspecific aspect, the invention provides an FCGR2 probe capable ofdetecting a target sequence as set out in Table 2C. In anotherembodiment, the invention provides an FCGR3 probe capable of detecting atarget sequence as shown Table 2D. Also provided is a control probewhich detects a target sequence of those of Table 2E. Furthermore, thereis provided the use of at least one of the target sequences depicted inTables 2C, 2D and 2E in a method for determining a CNP or a SNP in theFcγRII/FcγRIII gene cluster.

It is also possible to detect aberrant amounts of either activatingand/or inhibitory receptors at the transcriptional, i.e. mRNA level.Still further, gene product analysis can be performed at the proteinlevel using specific probes for the individual FcγR gene products, suchas MoAbs or (affinity-purified) polyclonal antibodies. However, asindicated above, antibodies described thus far cannot discriminatebetween the extracellular domains of the different members of the genecluster. Therefore, in the absence of specific reagents that candiscriminate between the different gene products at the protein level,analysis at the protein level is preferably accompanied by analysis atthe genetic (DNA and/or mRNA) level.

Conventional nucleic acid (DNA, RNA) and protein analytical methods canbe used to detect the amount of an FcγRII or RIII gene product accordingto the invention. For a review on quantitative analysis of nucleicacids, see Ding and Cantor (2004), J. Biochem. Mol.Biol. Vol 37, No. 1,pp. 1-10. (Q−) PCR analysis is particularly convenient for the purposeof the present invention. Preferred PCR techniques include LightCyclerand MPLA technology.

Only for the purpose of illustration, the method of the invention isexemplified by the analysis of material obtained from two types ofpatients, each suffering from a distinct autoimmune disease. However, itwill be clear for the skilled person that the underlying concept of thepresent invention is applicable to any type of autoimmune disease.

A specific aspect relates to a method for determining whether a subjectis predisposed for developing ITP, comprising determining in a sampleisolated from said subject the amount of genes, or gene productsthereof, of the FcγRII/FcγRIII gene cluster, said FcγRII/FcγRIII genecluster comprising the FCGR2C, FCGR3A, FCGR2A and FCGR3B genes encodingan activating FcγR, and FCGR2B encoding an inhibitory FcγR; andcorrelating said amount to the amount observed in a healthy population;wherein an increased amount of genes encoding an activating FcγR, orgene products thereof, and/or a decreased amount of gene encoding aninhibitory FcγR, or gene product thereof, is indicative of having anincreased chance of developing ITP. Preferably, the method comprisesdetecting the amount of the FCGR2C-ORF gene (product), determining theallele frequencies of the FCGR3A SNP for the FcγRIIIa-158F and/orFcγRIIIa-158V variant(s), and/or determining the allele frequency forthe promoter polymorphism −386G/C. More preferably, it comprises atleast determining the presence of the FCGR2C-ORF gene or gene product,e.g. messenger RNA or the FcγRII protein.

Another specific aspect relates to a method for determining whether asubject is predisposed for developing Kawasaki Disease (KD), comprisingdetermining in a sample isolated from said subject the amount of genes,or gene products thereof, of the FcγRII/FcγRIII gene cluster, saidFcγRII/FcγRIII gene cluster comprising the FCGR2C, FCGR3A, FCGR2A andFCGR3B genes encoding an activating FcγR, and FCGR2B encoding aninhibitory FcγR; and correlating said amount to the amount observed in ahealthy population; wherein an increased amount of genes encoding anactivating FcγR, or gene products thereof, and/or a decreased amount ofgene encoding an inhibitory FcγR, or gene product thereof, is indicativeof having an increased chance of developing KD.

Variation in FCGR2B exon-3 probe binding has never been observed in anyhealthy control or members of several multigeneration families testedfor inheritance patterns within the FCGR gene cluster. Preferably, themethod comprises detecting the amount of FCGR2B, for example bydetection of gain or loss of FCGR2B exon-3 amplification product, theFCGR2C-ORF gene (product), and/or the amount of FCGR3A (e.g. by CNPanalysis) More preferably, a method for determining the predispositionfor KD comprises at least determining the presence of FCGR2B exon-3probe binding and the FCGR2C-ORF gene or gene product, e.g. messengerRNA or the FcγRIIb and/or FcγRIIc protein.

In a still further aspect, the invention relates to the management ofautoimmune diseases. Most autoimmune diseases have a high morbidity,with crippling results for the patients, and cause a high incidence ofmortality. The quality of life of these patients is seriouslydiminished, resulting in high socio-economical and health costs.Treatment may consist of symptomatic treatment such as hormonesubstitution in case of endocrinological gland disease orimmunosuppressive therapy in case of more wide-spread disease tosuppress progression of disease and (multi)organ dysfunction. The majordrawback of general immunosuppression consists of a reduction of thehost's defence mechanisms against various infectious agents (evenresulting in opportunistic infections) and an increased risk ofmalignancies.

For some autoimmune diseases, treatment with intravenous immunoglobulin(IVIg) preparations or so-called IgG-based treatment consisting ofmurine MoAbs, humanized MoAbs, or (recombinant) human MoAbs or chimericmolecules with an Fc region to interact with any of the FcγRs(collectively referred to as ‘biologicals’) is the standard treatment ofchoice.

These preparations have been found to modulate the autoimmune phenomenawithout these afore-mentioned disadvantages. The use of high-dose IVIgfor the treatment of immune thrombocytopenic purpura (ITP) was firstreported more than two decades ago. After the therapeutic benefit ofIVIg was established in ITP, it was then successfully used to treat manyother autoimmune diseases. Although a complete definition of themechanism of IVIg action is still lacking, extensive research suggeststhat IVIg may achieve its therapeutic effects through multiplemechanisms. IVIg exerts immunomodulatory effects that may includeantiidiotypic neutralization of antiplatelet antibodies, stimulation ofFcγRIIb expression, and inhibition of FcγR-mediated platelet destruction(Jin and Balthasar, Hum Immunol. 2005; 66:403-10).

As with the other treatment modalities, IVIg works for many, but not forall patients. The increasing number of reports of IVIg for the treatmentof various autoimmune diseases and the general good clinical outcome ofthis therapeutic modality strongly indicate that IVIg has a role inthese conditions. Hence, it is important to identify those patients thatwould benefit most from this treatment.

The present inventors observed a correlation between the amount ofcertain FcγRII/FcγRIII gene cluster products and the responsiveness toIVIg therapy or treatment with biologicals. For example, an increasedamount of activating FcγR gene product, such as the presence ofFCGR2C-ORF, and/or a decreased amount of inhibitory FcγRIIb gene productwas found to be indicative of a poor response to IVIg therapy.Accordingly, the invention also provides a method to predict theresponsiveness of a patient suffering from an autoimmune disease totherapy with intravenous immunoglobulin (IVIg) or a monospecificbiological, such as a humanized monoclonal antibody or a chimericmolecule, comprising the C-terminal Fc-tail of IgG, comprisingdetermining in a sample isolated from said subject the amount of intactgenes, or gene products thereof, of the FcγRII/FcγRIII gene cluster,said FcγRII/FcγRIII gene cluster comprising the FCGR2C, FCGR3A, FCGR2Aand FCGR3B genes encoding an activating FcγR, and FCGR2B encoding aninhibitory FcγR; and correlating said amount to the amount observed in ahealthy population; wherein an increased amount of genes encoding anactivating FcγR, or gene products thereof, and/or a decreased amount ofgene encoding an inhibitory FcγR, or gene product thereof, is indicativeof having a reduced chance of being responsive to said therapy.

Thus, provided is a method to predict the responsiveness of a patient toIVIg therapy, comprising the determination of the amount of genes, orgene products thereof, of the FcγRII/FcγRIII gene cluster, saidFcγRII/FcγRIII gene cluster comprising the FCGR2C, FCGR3A, FCGR2A andFCGR3B genes encoding an activating FcγR, and FCGR2B encoding aninhibitory FcγR; and correlating said amount to the amount observed in ahealthy population; wherein an increased amount of genes encoding anactivating FcγR, or gene products thereof, and/or a decreased amount ofgene encoding an inhibitory FcγR, or gene product thereof, is indicativeof having a reduced chance of being responsive to said therapy.

Similar to what is described above, the analysis of the gene can be doneat the genomic, or at the corresponding transcriptional and/or thetranslational level. Also, the various above-mentioned combinations ofgenes and/or gene products can be detected. In one embodiment, theresponsiveness to IVIg therapy or to monospecific biologicals ispredicted by determining the amount of intact FCGR2C gene or geneproduct, preferably FCGR2C-ORF. Since the FcγRIIc is not expressed as afunctional activating receptor in the majority of a healthy populationdue to the homozygosity for the pseudogene of FCGR2C (containing theFCGR2C^(stop/stop) SNP), the presence of an intact FCGR2C-ORF gene willshift the balance to a relative excess of stimulatory signals. Becauseof the very high degree of homology between the extracellular domains ofall receptors within the FcγRII/FcγRIII gene cluster, these receptorscan at present not all be distinguished from each other by means ofdetection with MoAbs or polyclonal antibodies. As a consequence, anantibody that is used e.g. therapeutically to provide inhibitory signalsvia the inhibitory FcγRIIb receptor will also bind to the extracellulardomain of the activating FCGR2C-ORF gene product, FcγRIIc. Binding toand activation of the FcγRIIc receptor by the therapeutic compound willnot only counteract the effect mediated by FcγRIIb but it may even causea net increase in activating signals, thereby worsening the diseasecondition. Thus, in one embodiment the invention provides a method toidentify a subject that is likely to react negatively to therapy withIVIg therapy or biologicals, comprising detecting the presence orabsence of the FCGR2C-ORF gene or gene product. Preferably, it isdetected at the genetic level, optionally in combination with detectionat the protein level, for example using an antibody capable ofrecognizing FcγRIIc protein. Example 4 herein below exemplifies how theFCGR2C-ORF gene may be detected.

In a particular aspect, predicting IVIg/biologicals therapyresponsiveness comprises determining the copy number of said gene, forexample using MPLA technology. MPLA probes provided herein are suitablyused.

The patient to be considered for IVIg therapy is for example a subject,preferably a human child, diagnosed with Kawasaki Disease. Otherpatients that would benefit from the predictive method include patientswith Idiopathic Thrompocytopenic Purpura, Systemic Lupus Erythematosusor Rheumatoid Arthritis.

Experimental Section

Fc receptors are proteins on the surface of all leukocytes and plateletsthat are able to bind immunoglobulins, especially when these arecomplexed with antigens in an immune complex. Fc-gamma receptors (FcγR)specifically bind immunoglobulin G (IgG). FcγR exist in a number ofisoforms, namely FcγR type I, type II and type III. FcγRI is a receptorwith a high affinity for IgG, whereas FcγRII and FcγRIII have a loweraffinity. The distribution of these receptors over the various types ofleukocytes is shown in Table 1.

TABLE 1 FcγR diversity and cell distribution Subclass genes cellulardistribution motif FcγRI FCGR1 monocytes, macrophages, ITAM in γ-chainsIFNγ- or G(M)-CSF- treated neutrophils FcγRIIa FCGE2A monocytes,macrophages, ITAM in FcγR neutrophils, B lymphocytes and plateletsFcγRIIb FCGR2B B lymphocytes (macrophages, ITIM in FcγR neutrophils)FcγRIIc FCGE2C ??? ITAM in FcγR FcγRIIIa FCGR3A NK cells, macrophagesITAM in γ chains FcγRIIIb FCGR3B neutrophils, IFNγ- ITAM in γ chainstreated eosinophils

Binding of IgG (in a complex) to the FcγRs in general induces cellactivation, as, for instance, uptake of the immune complex, release ofcytotoxic proteins and generation of reactive oxygen species byphagocytic leukocytes (neutrophils, eosinophils andmonocytes/macrophages). Also the immunoglobulin production, the cytokineproduction and the cytotoxic reactions of specific subpopulation oflymphocytes are induced by Fcγ receptors. This cell activation ismediated by a so-called immunoreceptor tyrosine-based activation motif(ITAM) located in the cytoplasmic tail of the FcγR itself or incytoplasmic regions of associated proteins, which becomes phosphorylatedafter crosslinking of FcγR's on the cell surface by the immune complex.However, there is also an FcγR that dampens the immune response. ThisFcγRIIb contains an inhibitory ITIM motif in its cytoplasmic tail, whichassociates with tyrosine phosphatases that counteract thephosphorylation of the ITAM motif. Thus, a balanced immune response ismediated by an equilibrium between signals from the activating FcγRs andthe inhibiting FcγRIIb.

Recently, the importance of the inhibitory FcγRIIb has become clear fromstudies with knock-out mice. These mice, which lack the FcγRIIb, weresubjected to treatment that induces inflammatory reactions in the skin,the lung, the joints or the kidney. It turned out that the knock-outmice developed excessive auto-immune and chronic inflammatory reactions,which clearly proves that dampening of the immune response by signalsfrom FcγRIIb is indispensable for a normal physiologic reaction to anantigenic challenge (Clynes et al., J. Exp. Med. 1999; 189: 179; Yuasaet al., J. Exp. Med. 1999; 189: 187; Bolland & Ravetch, Immunity 2000;13: 277). For that reason, patients with the auto-immune disease SLEhave been investigated for the presence of SNPs in their FcγRIIb geneFCGR2B. However, until now, no clear correlation was found between theoccurrence of certain polymorphisms and the susceptibility to thisdisease.

Cytoband 1q21-23 is one of the regions implicated in susceptibility tomultiple autoimmune diseases. The FcγRII/III genes are located at 1q23,and a new family of genes, Fc receptor-like genes (FCRLs, also known asFcRHs), clusters nearby at 1q21 (Davis et al., 2001, 2002). FCRLs havehigh structural homology with the classical genes encoding the FcγRs,although the ligands and function of the FcR-like proteins are not yetknown. Region 1q23 is a candidate locus for susceptibility to SLE, andvariants in the classical FcγR II/III genes partially account fordisease susceptibility (Tsao, 2003; Ravetch and Bolland., 2001; Kyogokuet al., 2002). Although 1q21-23 is a good candidate region forcontaining rheumatoid arthritis-susceptibility genes, the association ofclassical FcγRs with disease susceptibility remains controversial (Nietoet al., 2001; Radstake et al., 2003).

We studied genes in the FcγR cluster and changes therein in detail inKawasaki disease, a major autoinflammatory disease, and ITP, anautoimmune hematological disease.

Kawasaki disease is an acute febrile syndrome in infancy, which ischaracterized by a vasculitis of mainly the medium-sized arteries. Sincethere is no specific test to diagnose the disease, diagnosis is made onclinical criteria. First, fever must be present for more than five days,not responding to antibiotics or antipyretics. Furthermore, at leastfour of the five following symptoms must be present: lymphadenopathy,edema and erythema of hand and feet, a polymorphous rash, bilateralconjunctivitis, enanthema and inflammation of the mucous membranes(Kawasaki, 1979, Burns & Glode, 2004). The incidence of Kawasaki diseaseis 5-17 per 100 000 in Europe and the United States, mostly in childrenunder the age of five years. For unknown reasons the incidence in Japanis 135 per 100 000. In approximately 25% of the children the vasculitiswill lead to coronary aneurysmatic lesions (CAL) as detected byechocardiography, which may develop during the acute stage of thedisease when untreated. This makes it the leading cause of acquiredheart disease in children. The coronary vasculitis may cause death bysudden rupture when becoming giant in diameter, or cause flowdisturbance and thrombosis within the aneurysmatic lesions, resulting inacute myocardial infarction. Following recovery, Kawasaki disease mayeventually lead to heart disease in adolescence or young adulthood dueto progressive stenosis and calcification of affected coronary arteries.

Therapy consists of a single high-dose of IVIg (2 g/kg) infused in 8-12hours and aspirin, resulting in a decrease of complications to 5-16%(Newburger et al., 1991, Tse et al., 2002). To date little is knownabout the working mechanisms of IVIg and why this therapy is successfulin many and fails in other patients with Kawasaki disease, especially inthe very young. Inadequate handling or insufficient clearance ofmicrobial antigens by IgG-dependent mechanisms might, in certainindividuals, be responsible for this difference.

ITP patients suffer from a bleeding tendency. The low number ofplatelets in the circulation of these patients is caused by the presenceof auto-antibodies against platelets. Standard therapy is again IVIg orcorticosteroids.

Systemic lupus erythematosus (SLE) is the archetypal autoimmune diseasegiven its complex clinical and molecular manifestations. Like the otherrheumatic diseases, appropriate management is critically dependent uponthe proper assessment of disease activity, organ damage, and quality oflife. SLE patients suffer from an auto-immune disease characterized bythe production of antinuclear auto-antibodies caused by a failure of Bcell tolerance and tissue deposition of immune complexes. Thus, the Blymphocyte plays a key role in SLE pathogenesis by bothautoantibody-dependent and autoantibody-independent mechanisms.Additionally, aberrant interactions between B and T cells are criticalto disease emergence and progression. New agents that directly targetimmune cells abnormal in SLE include the B-cell depleting or modulatingantibodies, rituximab (anti-CD20) and epratuzumab (anti-CD22) and theanti-dsDNA tolerogen LJP394. Immune cells can also be manipulatedindirectly through cytokine effects. For B cells, anti-BAFF (B-cellactivation factor of the tumor necrosis family) provides an example ofthis approach. Other, more pleiotropic cytokines can likewise be blockedin SLE. In addition to the blockade of interleukin-10 (IL-10), the firstanti-cytokine approach examined, it is mainly anti-TNF therapy that hascome into focus, holding promise for some patients with lupus nephritis.Many additional cytokines, such as IL-6, IL-18, and the type Iinterferons, represent interesting future targets in which antagonismdepend on neutralizing properties as well as FcγR-mediated effects.

Rheumatoid arthritis, juvenile idiopathic arthritis, the seronegativespondyloarthropathies including psoriatic arthritis, and SLE are allexamples of rheumatic diseases in which inflammation is associated withskeletal pathology. Although some of the mechanisms of skeletalremodeling are shared among these diseases, each disease has a uniqueimpact on articular bone or on the axial or appendicular skeleton. Theblockade of TNF by so-called biologicals (anti-TNF humanized murinemonoclonal antibodies or chimeric molecules engineered as N-terminalbinding domains derived from authentic receptors and C-terminalFcγR-binding Fc-tail) with strong TNF-binding properties havesignificant impact on the therapy of a number of chronic autoimmunediseases, such as the aforementioned rheumatic diseases as well asinflammatory bowel disease. The experience with this therapy inrheumatoid arthritis (RA) and Crohn's disease (CD) have spread itsapplication into other autoimmune diseases, and has led to augmentedtherapeutic benefit. Other promising approach has been to blockco-stimulatory interactions between T and B cells in rheumatic diseases,for example by inhibiting the CD40-CD40 ligand pathway with anti-CD40ligand monoclonal antibody or the B7 pathway with the chimera CTLA4-Ig.There are still many limitations, but the prospects for the future ofsuch biologicals is intriguing. The Fcγ-binding properties of thesebiologicals make the therapeutic efficacy subject to the same influenceof FcγRs as demonstrated in case of IVIg via FcγR-mediated activities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Location of MLPA probes within the FCGR2 gene cluster (panel A)and the FCGR3 gene cluster (panel B). Several probes within eachseparate gene can be used to determine the CNPs and the SNPs. Note thatthe probe that recognizes FCGR3B-NA1 is the same that recognizes FCGR3A.

FIG. 2. Distribution of mRNA ratios of Fc-γRIIa and Fc-γRIIb in fiftyhealthy Caucasian individuals.

FIG. 3: FcγRIIc expression and function.

-   -   A. Distribution of FcγRIIc mRNA expression on leucocytes.    -   B. FcγRII expression on NK cells is limited to the FCGR2C-ORF        genotype. Blood cells were incubated with CD56 and CD32.        Lymphocytes were gated on basis of their forward scatter/side        scatter pattern. NK cell population was determined as        CD56-positive lymphocytes.    -   C. FcγRIIc expression on NK cells is modulated by IL-15 but not        by IL-2. PBMCs were isolated and cultured. Expression on CD56⁺        NK cells was measured by flow cytometry with CD32 at the        indicated time-points. There was a significant higher expression        on day 2 and 5 on the IL-15 stimulated NK cells (p=0.01, n=5)    -   D. FcγRIIc mRNA is strongly upregulated by GM-CSF on cells of        FCGR2C-ORF donors. Neutrophils and PBMCs were isolated and        cultured for 4 hrs with the indicated stimuli. FcγRIIc mRNA was        measured by quantitative RT-PCR. GM-CSF strongly upregulated        FcγRIIc mRNA in neutrophils and to a lesser extent in PBMCs of        FCGR2C-ORF genotyped donors, but not in FCGR2C-Stop donors        (p=0.0001 and p=0.01, respectively; n=5-8).    -   E. rADCC. PBL were isolated and FcγRIIc functionality was        assessed by rADCC. Cells from both FCGR2C-Stop and FCGR2C-ORF        genotyped donors killed anti-FcγRIII-coated targets with similar        kinetics (upper left and lower left panels, respectively). In        contrast, only cells from FCGR2C-ORF genotyped donors were        capable of killing anti-FcγRII coated targets (right lower        panel) (n=4).    -   F. rADCC with stimulated cells. PBL were obtained and        subsequently cultured for 2 days with or without IL-2 or IL-15.        Thereafter, the cells were harvested and used in a rADCC. Cells        from both FCGR2C-Stop and FCGR2C-ORF genotyped donors killed        anti-FcγRIII-coated targets (left panel). In both cases IL-15        and to a lesser extent IL-2, enhanced specific lysis of the        anti-FcγRIII-coated targets (n=3). In contrast, only cells from        FCGR2C-ORF genotyped donors were capable of killing anti-FcγRII        coated targets (right panel). Here, again IL-15 strongly        enhanced the specific cell lysis (n=3).

EXAMPLES Example 1 Validation CYBB Gene as Reference Gene

Primer Sequences:

Albumin Alb-F primer 5′-TGAAACATACGTTCCCAAAGAGTTT-3′ (SEQ ID NO: 1)Alb-R primer 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′ (SEQ ID NO: 2) FCGR2BFCGR2Bex3-(Eco I)-F 5′-CTCTCAAGCTCCTTGGGCTTCCTCTTCT-3′ (SEQ ID NO: 3)FCGR2Bex3-(Not I)-R 5′-ACCCCCCTTTCCTGCAAACCTCTGC-3′ (SEQ ID NO: 4)Y-chrom SRY 109F 5′-TGGCGATTAAGTCAAATTCGC-3′ (SEQ ID NO: 5) SRY 245R5′-CCCCCTAGTACCCTGACAATGTATT-3′ (SEQ ID NO: 6) CYBB CYBBex8-(HindIII)-F5′-GAGATCAAGCTTATGTCAAATATTTAA (SEQ ID NO: 7) GCAAGCCTAC-3′CYBBex8-(XhoI)-R 5′-GAGATCCTCGAGACTTGTCCATGATATA (SEQ ID NO: 8)GTTAGACAC-3′FCGR2B/ALB Construct

To quantify the FCGR2B copy number with quantitative RT-PCR, a constructwas made containing an 80-bp fragment of the ALB gene and 400-bpfragment of FCGR2B by Baseclear Labservices BV. For detailed protocol werefer to BaseClear Labservices B.V, project code 11926.

In brief, a PCR was performed on a human DNA sample to obtain a PCRproduct of the ALB gene fragment using primers Alb-F and Alb-R, andprimer pair FCGR2B-ex3-F and FCGR2B-ex3-R to obtain the FCGR2B exon 3fragment. PCR products were blunted and phosphorylated (one of theproducts). Products were ligated overnight and a PCR was set up (on theligation product) in order to amplify the total 480-bp fragment. PCRproducts were cloned into a pGEMT-easy vector, clones were picked andsequenced to confirm the sequence.

FCGR2B/CYBB Construct

Because a variation in the ALB gene copy number was observed, a newconstruct was made with CYBB as a reference/calibrator gene. Theconstruct is based on a plasmid containing a 217-bp fragment of the CYBBgene and the 400-bp fragment of the FCGR2B gene. For this construct aBluescript SK +/−phagemid was used. Using a standard cloning protocol,the CYBB fragment was cloned into the Bluescript SK +/−phagemid usingthe restriction enzymes HindIII and XhoI. Clones were picked andsequenced to confirm the inserted CYBB sequence. Subsequently the FCGR2Bfragment was cloned in by using restriction enzymes EcoRI and NotI.Again the clones were sequenced to confirm the inserted sequences. Withthe restriction enzyme ScaI the plasmid was made linear. The constructcontaining the CYBB fragment can also be used in combination with othertarget genes or fragments thereof (e.g. FCGR2A, FCGR2C).

Quantitative Real-Time PCR

Amplification by PCR was performed on a LightCycler instrument (Roche,Almere, The Netherlands), with software version 3.5. The reaction wasperformed with Lightcycler FastStart DNA Master^(PLUS) SYBR Green I(Roche). The annealing temperature for the ALB and FCGR2B primers was65° C., for the CYBB primers 60° C. The reaction mixture consisted of 4μl of the plasmid or 4 μl (100 ng) of genomic DNA, 1 μl of the forwardand 1 μl of the reverse primer (1 μM), 4 μl of LightCycler FastStart DNAMasterPLUS SYBR Green I mix (Roche) and 10 μl of water. The PCR startswith a pre-incubation step for 10 min at 95° C. followed by 40 cycles ofdenaturation of 5 s at 95° C., annealing of the primers at 60° C. or 65°C. depending on the primers used for 30 s, followed by extension at 72°C. for 15 s. At the end of 40 cycles, a melting curve was generated todetermine the specificity of the amplified products. Either a 10-foldserial dilution of the construct containing the FCGR2B/ALB or theconstruct containing the FCGR2B/CYBB (1:1) was used to construct astandard curve to which each sample was quantified. The threshold cycle(Ct) values of the serial diluted construct samples, determined by the‘second derivative maximum method’ were used to calculate and plot alinear regression curve, as performed by the software. From thisregression, the quality of the standard curve can be evaluated by theslope and correlation coefficient (r). The slope of the curve was usedto calculate the efficiency of the PCR reaction (E). The E from thedifferent PCR primer combinations did not differ more than 0.05. The 4points of the standard curve were assigned a dimensionless quantity of1000, 100, 10 and 1, respectively. Subsequently, the software can assigna dimensionless quantity to the unknown samples depending on theirobserved Ct value. Unknown samples were run in duplicate. For eachsample the calculated value, depending on the Ct value, of the FCGR2Bwas divided by the value calculated for the reference/calibrator geneused in the assay (either albumin or CYBB).

When the FCGR2B/ALB construct was used for the standard curve, anobserved outcome of an unknown sample (FCGR2B amount/ALB amount) of 1means the individual is carrying equal amounts of copy number FCGR2Bcompared to ALB (thus far generally believed to be 2 copies). As CYBB isan X-linked gene, when the FCGR2B/CYBB construct is used, theinterpretation of the outcome depends on the analyzed DNA being from amale or a female individual.

Results

From the first obtained results using the FCGR2B/ALB construct it becameclear that the ALB gene is not a good reference gene. Observed outcomevalues of unknown samples of for instance 0.25 would imply a copy numberof 0.5 FCGR2B. The inventors hypothesized that this result can only beobserved when an individual is carrying 1 copy of FCGR2B and 4 copies ofthe gene encoding albumin, or 2 copies of FCGR2B and 8 copies of ALB. Totest this hypothesis a quantitative RT-PCR was set-up with primersspecific for the Y-chromosome and the ALB gene. Serial 10-fold dilutionsfrom DNA from a male were used to construct a standard curve. For a malethe expected outcome would be 1 (amount product Y-chrom/amount productALB, as comparable to the standard curve used). However for theabove-mentioned sample an outcome of 0.5 was observed. Inventorsconcluded that this can only be explained by 4 copies of the gene foralbumin. To test this hypothesis further, also a quantitative PCR wasset-up with CYBB specific primers and ALB. Again a 10-fold serialdilution of a male was used to construct the standard curve. The resultfrom this test confirmed the earlier observed outcomes. Therefore, theconstruct containing FCGR2B/ALB was no longer used and a new constructcontaining FCGR2B/CYBB was developed.

The construct containing CYBB as a reference/calibrator gene was alsoused to make the following constructs: FCGR2A/CYBB, FCGR2C/CYBB.

Example 2 Copy Number Determination in Autoimmune Disease Patients UsingLightcycler Technology

Using the method described in example 1, 40 Kawasaki Disease (KD)patients and 40 age-matched healthy controls were analyzed. In 17 KDpatients a loss of copy number FCGR2B was observed, whereas this wasonly observed in 8 controls (p<0.05).

Example 3 Quantitative Reverse Transcriptase PCR for the Quantificationof Transcripts

mRNA was isolated from 10⁷ purified neutrophils and reverse transcribedinto cDNA. Intron-spanning primers were designed to specifically amplifycDNA and exclude amplification of genomic DNA, yielding products of 100bp for P-glucuronidase (GUS), 244 bp for FcγRIIa and 243 bp forFcγRIIb2.

Primer sequences:

β-glucuronidase: fw primer: 5′-GAAAATATGTGGTTGGAGAGCTCATT-3′,(SEQ ID NO: 9) (GUS) rev primer: 5′-CCGAGTGAAGATCCCCTTTTTA-3′.(SEQ ID NO: 10) FcγRIIa: fw primer: 5′-ATCATTGTGGCTGTGGTCATTGC-3′,(SEQ ID NO: 11) rev primer: 5′-TCAGGTAGATGTTTTTATCATCG-3′.(SEQ ID NO: 12) FcγRIIb2: fw primer: 5′-GGAAAAAGCGCATTTGAGCCAATC-3′,(SEQ ID NO: 13) rev primer: 5′-GGAAATACGAGATCTTCCCTCTCTG-3′.(SEQ ID NO: 14)Polymerase Chain Reaction

Amplification by PCR was performed on a LightCycler instrument (Roche,Almere, The Netherlands), with software version 3.5. The reaction wasperformed with LightCycler FastStart DNA Master^(PLUS) SYBR Green I(Roche), which has been optimized by the manufacturer such that MgCl₂optimalisation is no longer needed. The annealing temperature used forall primers was 60° C. The reaction mixture consisted of 2 μl of cDNA, 1mM of each primer combination and 4 μl of LightCycler FastStart DNAMaster^(PLUS) SYBR Green I mix (Roche) in a total volume of 20 μl. AllcDNA amplified was compared to the standard within the same run and inevery run the same standard was used, although there was very littlevariation in the standard between runs. For amplification the followingLightCycler protocol was used. The chemical cleft of the Taq polymerasewas removed by preincubation for 10 minutes at 95° C. to activate theTaq polymerase; the template was amplified for 40 cycles of denaturationof 5 seconds at 95° C., annealing of the primers at 60° C. for 30seconds, followed by extension at 72° C. for 15 seconds. The amount offluorescence was measured at the end of each cycle at 72° C. At the endof 40 cycles, a melting curve was generated to determine the uniquefeatures of the DNA amplified. To identify the product obtained, it wassubmitted to a 1%, w/v, agarose gel to determine the size. Subsequently,the band obtained was purified by means of GFX PCR DNA and Gel Bandpurification kit (Amersham Biosciences) according to the manufacturer'sinstructions to remove excess of dNTP's and primers. The product wassequenced by Big-dye Terminator Cycle Sequencing (version 1.1) and ABIPrism software (Applied Biosystems, Foster City, Calif., USA). Thesequence obtained was verified with BLAST(http://www.ncbi.nlm.nih.gov/BLAST/) to determine specificity. Allproducts obtained were unique and had no overlap with other isoforms.

Standard Curves and Relative Quantitation

As a source of cDNA for standard curves to which all samples werenormalized, neutrophils were isolated from an apheresis buffy coatobtained from the bloodbank North-West (Sanquin). Serial 10-folddilutions from the cDNA obtained were made to which each sample wasquantified with the method described in Technical Note No. LC 13/2001(Roche Applied Science). In short, the threshold cycle (C_(T)) values,determined by the LightCycler software, were used to calculate and plota linear regression curve, as performed by the software. From thisregression, the quality of the standard curve can be evaluated by theslope and the correlation coefficient (r). The slope of the line wasused to determine the efficiency of the reaction (E). From the C_(T)'Sand the efficiencies obtained, the normalized ratio can be calculatedwith the following formula: E_(T) ^(CpT(C)−CpT(S)):E_(R)^(CpR(C)−CpR(S)). In which E_(T) is the efficiency of the PCR of thetarget gene; ER the efficiency of the PCR of the reference gene; CpT(C)is the measured C_(T) of the target gene determined for standard orcalibrator; CpT(S) is the measured C_(T) of the target gene determinedfor the sample; CpR(C) is the measured C_(T) of the reference gene ofthe calibrator or standard; and CpR(S) is the measured C_(T) of thereference gene of the sample.

Validation of Quantitative RT-PCR for FcγRII Isoforms on the LightCycler

To study the expression levels of FcγRII isoforms on neutrophils, we setup a relative quantitative RT-PCR by means of the LightCyclerinstrument. This technique yielded a highly sensitive and specificmethod to determine FcγRIIa and FcγRIIb2 mRNA expression levels inneutrophils. The slopes of the standards for each PCR reaction werearound −3.3, yielding an efficiency of about 2, indicating that duringeach cycle the specific product was doubled. Furthermore, each set ofprimers used resulted in a specific melting curve with its own meltingtemperature (Tm), whereas the non-template controls displayed adifferent melting curve or no product at all. FIG. 2 shows that distinctneutrophil mRNA ratios for FcγRIIa:FcγRIIb were found in a normalCaucasian population.

Example 4 MLPA Assay with Newly Developed Probes

DNA was isolated with either the Puregene DNA isolation kit (Biozym,Hess, Oldendorf, Germany) or the QIAamp DNA Blood kit (Qiagen, Hilden,Germany). To analyse the FCGR gene cluster a Multiplex Ligation ProbeAmplification assay is used. New probes were designed that are highlyspecific for the FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B genes. As theFCGR gene cluster is very homologous, this was not possible for allsites. At least 3 probes per gene were designed to cover every singlegene. In this way CNPs and partial insertion/deletions can be studied.Separate probes were designed to study known (functional) SNPs in FCGR2A(131 H/R), FCGR2B (232 I/T), promotor of FCGR2B and C (−386, −120),FCGR3A (NA1/NA2/SH) and FCGR3B (158 V/F). For an overview of thelocation and the specific target sequences of the probes see FIGS. 1Aand 1B and Table 2 below. Probes were manufactured according to Schoutenet al., 2002. Because of the homology between certain probes, probeshave to be divided over two separate mixes (probe MIX 1 and probe MIX 2;see tables 2A and 2B, respectively) to prevent competition.

TABLE 2A “Probe MIX 1”. Length Probe # Gene/Chr. position Comments 1300797-L0463 Control probe, Chr. 5q31 136 2271-L2327 Control probe, Chr.1p36 142 3605-L2972 FCGR2A, exon 1 (D01A) 148 3610-L2977 FCGR2C, exon 7(D07) 154 2679-L2144 Control probe, Chr. 1q25 160 3616-L2983 FCGR3A,exon 3 (D03) SNP detects NA1 variant and FCGR3A 172 2677-L2143 Controlprobe, Chr. 1q25 181 3611-L2978 FCGR2B, exon 1 (D01A) Detects alsoFCGR2C 193 3606-L2973 FCGR2A, exon 1 (D01B) 202 4812-L4198 FCGR2B,232I/T (D05B) SNP detects the frequent 232-I variant 211 3609-L2976FCGR2C, exon 6 (D06) 220 3619-L2986 FCGR2B, intron 3 (D03) Atintron/exon boundary of exon 4 229 1487-L1095 Control probe, Chr. 16q24238 0974-L0561 Control probe, Chr. 14q12 265 1325-L0874 Control probe,Chr. 17p13 274 3613-L2980 FCGR2B, exon 6 (D06A) 283 3608-L2975 FCGR2A,exon 5 (D05A) 292 3162-L2603 Control probe, Chr. 11p11 301 1916-L1460Control probe, Chr. 1q21 310 3618-L2985 FCGR3B, exon 5 (D05) 3193612-L2979 FCGR2B, exon 7 (D07A) 328 1918-L1462 Control probe, Chr. 1q21337 3614-L2981 FCGR3A, exon 1 (D01) 346 3615-L2982 FCGR3B, exon 1 (D01)355 4813-L4187 FCGR2A, 131H/R (D04A) SNP detects 131-H variant 3732560-L2023 Control probe, Chr. 3q23 382 2908-L2302 Control probe, Chr.Xq23 391 4816-L4190 FCGR3A, 158V/F (D04) SNP detects 158-F variant 4004818-L4192 FCGR2C, 2C-STOP-specific Probes in bold indicate those whichare required for a reliable assay outcome. Target sequences of theprobes are presented in Tables 2C, D and E.

TABLE 2B “Probe MIX 2”. Length Probe # Gene/Chr. position Comments 1300797-L0463 Control probe, Chr. 5q31 136 2271-L2327 Control probe, Chr.1p36 142 4817-L4191 FCGR2B, exon 3 (D03B) Detects FCGR2B and FCGR2C-ORF148 3610-L2989 FCGR2A, exon 7 (D07) 154 2679-L2144 Control probe, Chr.1q25 165 3616-L2990 FCGR3B exon 3 (D03) SNP detects NA2 variant 1722677-L2143 Control probe, Chr. 1q25 181 3611-L2978 FCGR2B, exon 1 (D01A)Detects FCGR2B and FCGR2C 202 4812-L4186 FCGR2B, 232I/T (D05B) SNPdetects rare 232-T variant 211 4815-L4189 FCGR2A, exon 6 (D06B) 2203619-L2994 FCGR2C, intron 3 (D03) At intron/exon boundary of exon 4 2291487-L1095 Control probe, Chr. 16q24 238 0974-L0561 Control probe, Chr.14q12 247 4821-L4195 FCGR2A, exon 3 (D03C) 265 1325-L0874 Control probe,Chr. 17p13 274 3613-L2980 FCGR2B, exon 6 (D06A) 283 4819-L4193 FCGR3B,SH (D03SH) SNP FCGR3*3 (SH) variant 292 3162-L2603 Control probe, Chr.11p11 301 1916-L1460 Control probe, Chr. 1q21 310 3618-L2993 FCGR3A,exon 5 (D03) 319 3612-L2979 FCGR2B, exon 7 (D07A) 328 1918-L1462 Controlprobe, Chr. 1q21 355 4814-L4188 FCGR2A, 131 H/R (D04B) SNP detects 131-Rvariant 373 2560-L2023 Control probe, Chr. 3q23 382 2908-L2302 Controlprobe, Chr. Xq23 391 4816-L4196 FCGR3A, 158V/F (D04) SNP detects 158-Vvariant and FCGR3B Probes in bold indicate those which are required fora reliable assay outcome. Target sequences of the probes are presentedin Tables 2C, D and E.

TABLE 2C Sequence information of FCGR2 probes used Length Mix1 . . .. . . Mix2 Probe # Gene # Sequence detected 142 3605-L2972 FCGR2A-D01AGTCTCAGAATGTATGTCCCAGAAACCTGTGGCTGCTTCAA (SEQ ID NO: 15) . . . 1933606-L2973 FCGR2A-D01B ATAGTCATCCCAGCACTGTGCCAACGTCCAGTGGGTTTTA(SEQ ID NO: 16) . . . 247 4821-L4195 FCGR2A-D03CTCTGTGACTCTGACATGCCAGGGGGCTCGCAGCCCTGAGA (SEQ ID NO: 17) 355 4813-L4187FCGR2A-D04A AAATCCCAGAAATTCTCCCATTTGGATCCCACCTTCTCCAT (SEQ ID NO: 18). . . 355 4814-L4188 FCGR2A-D04BGGAGAAGGTGGGATCCAAACGGGAGAATTTCTGGGATTTT (SEQ ID NO: 19) 283 3608-L2975FCGR2A-D05A TCATTGCGACTGCTGTAGCAGCCATTGTTGCTGCTGTAGT (SEQ ID NO: 20). . . 211 4815-L4189 FCGR2A-D06BAGATGGCTGGGATTACTCACCTCAAATTGGGCAGCCTTCA (SEQ ID NO: 21) . . . 1813611-L2978 FCGR2B-D01A ATAGCACAGCTGTCCACAGAAGCATATGACCCCAAGGCTG(SEQ ID NO: 22) 220 3619-L2986 FCGR2B-D03CTGAAAGACACAAAAAACCGCAGAGGACCCGGGAGGTCTT (SEQ ID NO: 23) . . . 2203619-L2994 FCGR2B-D03 GTGAAAGACACAGAAAACCCCAGAGGACCCGGGAGGTCTT(SEQ ID NO: 24) . . . 142 4817-L4191 FCGR2B-D03BGGCAGAGAGAGGAGGTAGCATGAAGAAGAGGAAGCCCAGG (SEQ ID NO: 25) . . . 2024812-L4186 FCGR2B-D05B-C GTGGCTGTGGTCACTGGGACTGCTGTAGCGGCCATTGTTG(SEQ ID NO: 26) 202 4812-L4198 FCGR2B-D05B-TGTGGCTGTGGTCACTGGGATTGCTGTAGCGGCCATTGTTG (SEQ ID NO: 27) . . . 2743613-L2980 FCGR2B-D06A CAATCCCACTAATCCTGATGAGGCTGACAAAGTTGGGGTG(SEQ ID NO: 28) . . . 319 3612-L2979 FCGR2B-D07ACAATCACCTATTCACTTCTCATGCACCCGGATGCTCTGGA (SEQ ID NO: 29) 211 3609-L2976FCGR2C-D06 GAAGGCTGCCCAATTTGAGATGAGTAATCCCAGCCATCTC (SEQ ID NO: 30). . . 148 3610-L2989 FCGR2A-D07 GCCATCAGAAAGAGACAACTTGAAGAAACCAACAATGACT(SEQ ID NO: 31) 148 3610-L2977 FCGR2C-D07GCCATCAGAAAGAGACAACCTGAAGAAACCAACAATGACT (SEQ ID NO: 32) 400 4818-L4192FCGR2C-STOP TTGGGCTTCCTCTTCCTCACGCTACCTCCTCTCTCTGCCC (SEQ ID NO: 33)

TABLE 2D Sequence information of FCGR3 probes used Length Mix1 . . .. . . Mix 2 Probe # Gene # Sequence detected 337 3614-L2981 FCGR3A-D01TCCAGGCTCTTTCCTTCCTGGTCCTGTTCTATGGTGGGGC (SEQ ID NO: 34) 160 3616-L2983FCGR3A-D03 CTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCT (SEQ ID NO: 35) 3914816-L4190 FCGR3A-D04 CTACTTCTGCAGGGGGCTTTTTGGGAGTAAAAATGTGTCT(SEQ ID NO: 36) . . . 391 4816-L4196 FCGR3A-D04CTACTTCTGCAGGGGGCTTGTTGGGAGTAAAAATGTGTCT (SEQ ID NO: 37) . . . 3103618-L2993 FCGR3A-D05 AGACTGGAAGGACCATAAATTTAAATGGAGAAAGGACCCT(SEQ ID NO: 38) 346 3615-L2982 FCGR3B-D01TCCACCCATCTCTGTCACCTGCCAGTTTCCTTTTCTTGAA (SEQ ID NO: 39) . . . 1653616-L2990 FCGR3B-D03 CTCAATGGTACAGCGTGCTTGAGAAGGACAGTGTGACTCT(SEQ ID NO: 40) 310 3618-L2985 FCGR3B-D05AGACTGGAAGGACCATAAACTTAAATGGAGAAAGGACCCT (SEQ ID NO: 41)

TABLE 2E Sequence information of Control probes used LengthMix1 and Mix2 Probe # Gene/# Sequence detected 130 0797-L0463 IL4-D01CACTGCAAATCGACACCTATTAATGGGTCTCACCTCCCAA (SEQ ID NO: 42) 136 2271-L2327CAB45-D01 CCACCAGGAGGTCTTCCTAGGCAAGGACCTGGGTGGCTTT (SEQ ID NO: 43) 1542679-L2144 HRPT2-D11 GAGGGTGCATCTGCCCGGAAGACTCAGACTCCTGCAGCCC(SEQ ID NO: 44) 229 14871-L1095 FANCA-D15AAGAAGGCCCTGGTCTTCCTGTTTACGTTCTTGTCAGAAC (SEQ ID NO: 45) 238 0974-L0561TINF2-D01 GCGGACGCTGCGTGGAACATTTTCCGCGAGTACTGGAGTT (SEQ ID NO: 46) 2651325-L0874 ASPA-D05 GCCATTGAGGTCTATAAAATTATAGAGAAAGTTGATTACC(SEQ ID NO: 47) 292 3162-L2603 MNYBPC3-D29CGCTTCCGCATGTTCAGCAAGCAGGGAGTGTTGACTCTGG (SEQ ID NO: 48) 301 1916-L1460LMNA-D02 AGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCT (SEQ ID NO: 49) 3281918-L1462 LMNA-D04 AGACCCGACTGGTGGAGATTGACAATGGGAAGCAGAGTGA(SEQ ID NO: 50) 373 2560-L2023 ATR-D47AGACCCATGTTCTTGACATTGAGCAGCGACTACAAGGTGT (SEQ ID NO: 51) 382 2908-L2302PAK3-D01 GTAACAACCGGGATTCTTCAGCACTCAACCACAGCTCCAA (SEQ ID NO: 52)MLPA Assay

The MLPA assay was performed according to the instructions of themanufacturer (MLP Holland BV, Amsterdam, The Netherlands). In brief, 5μl of DNA (20 ng/μl) is denatured at 98° C. for 5 min and then cooled to25° C. in a thermal cycler with heated lid. 1.5 μl of probe mix and 1.5μl of buffer is added to each sample and incubated for 1 min at 95° C.followed by 16 hr at 60° C. Then 32 μl of Ligase-65 mix is added to eachsample while at 54° C., followed by an incubation of 15 min at 54° C.and 5 min at 98° C. This ligation mixture is diluted 4 times. 10 μl ofpolymerase mix is added containing one single primer pair. Directlyafter adding the polymerase mix the PCR reaction is started. PCRconditions are 36 cycles of 30 s 95° C., 30 s 60° C., 60 s 72° C.followed by 20 min at 72° C. Following the PCR reaction 1 μl of the PCRreaction is mixed with 0.5 μl CXR 60-400 (Promega) internal sizestandard and 8.5 μl of deionised formamide and incubated for 10 min at90° C. Products are then separated by electrophoresis on an ABI-3100.

Data Analysis

Analysis of a limited number of samples can be done by visualexamination of the capillary electrophoresis peak. For the analysis oflarge number of samples the program Genemarker (v 1.30) is suitablyused. In brief, the capillary electrophoresis peak profiles are importedinto the program and then signals are normalised for signal strengthusing the assigned control probes. As MLPA is a relative quantificationmanner one sample has to be assigned as reference sample. The normalizedheight (or the area) of the probe amplification product of the unknownsample is divided by the normalized height of that probe amplificationproduct of the reference. A ratio between 0.8-1.2 is considered normal,below 0.8 is considered as loss in copy number and above 1.2 as gain ofcopy number.

Results on Kawasaki Disease Patients

SNPs; By conventional SNP PCR no significant differences can be detectedin samples from 170 patients with Kawasaki disease (Biezeveld et al. Fcgamma Receptors in Kawasaki disease, The involvement of Fc gammareceptor gene polymorphisms in Kawasaki disease. Clin Exp Immunol. 2007;147: 106-111). DNA of 70 known samples (used in a previous study withconventional PCR methods to genotype the SNPs in the FCGR gene cluster)were analysed with the MLPA assay to validate the probes identifying theSNPs. The only discrepancies observed were caused by the fact that withthe conventional methods no absolute quantification is possible andtherefore a sample with only 1 FCGR3B allele is typed as NA2/NA2(homozygous) instead of NA2/null with the new MLPA method.

CNPs; DNA of 70 Kawasaki Disease (KD) patients and 100 healthy Caucasiancontrols was tested.

-   -   in 30 KD samples an altered FCGR2B exon-3 amplification product        was observed, whereas this was observed in none of the control        samples.    -   in 23 KD (32%) samples a FCGR2C-ORF (open reading frame in        exon 3) resulting in an activating receptor was observed,        compared to 18% in the healthy controls (p<0.05).    -   in 3 KD samples 3 copies of the complete FCGR3A gene were        observed, not found in control samples.    -   in 3 KD samples and 2 controls samples 3 copies of the complete        FCGR3B gene were observed without the SH-variant being present        on one of the copies.        Results on ITP Patients

DNA of 116 samples of ITP patients were tested by the MLPA assay.Increased variation in the FCGR2 and FCGR3 gene clusters was found inCaucasian ITP patients as compared to healthy Caucasian controls(n=100).

-   -   In the FCGR2C gene a SNP in exon 3 converts a glutamine in the        open reading frame (ORF) to the most commonly found stop codon.        In the control group 82% of the individuals were homozygous        FCGR2C^(stop/stop). Whereas, the FCGR2C-ORF gene was present in        18% of healthy individuals, FCGR2C-ORF was found in 35% of the        patients with the hematological autoimmune disease ITP (n=116;        odds ratio (OR): 2.4, 95%-confidence interval (95% CI): 1.3-4.3;        p=0.009). We have evidence that the FCGR2C-ORF gene translates        into an activating IgG receptor on the cell membrane, exerting        antibody-mediated activity such as antibody-dependent cellular        cytotoxicity (ADCC) by immune cells.    -   Also a significant difference in genotype (p=0.01) and allele        (p=0.02) frequency for the promoter polymorphism −386G/C was        observed. The genotype −386CC was rare, and only observed in an        ITP patient.    -   In the same series of DNA samples, we confirmed the previously        observed overrepresentation of the SNP in FCGR3A encoding the        FcγRIIIa-158V variant, being most prevalent in ITP of        childhood-onset (p=0.0005) and not in ITP of adult-onset        (p=0.3).        FCGR2C Splice Variants.

To date, five splice variants of FcγRIIc have been reported, two ofwhich result in a membrane-anchored receptor, i.e. FcγRIIc1 encoded bythe full transcript, and FcγRIIc3 reported to lack exon 7. Wehypothesized that alternative splicing of the FcγRIIc transcript isrestricted to the FCGR2C-stop allele. To investigate this, we cloned theFcγRIIc transcripts from a single individual with an FCGR2C^(ORF/ORF)genotype and three individuals with an FCGR2C^(ORF/stop) genotype into abacterial expression vector and obtained the sequence of at least 20clones per individual. Upon sequencing of all these clones it was foundthat individuals genotyped as FCGR2C^(ORF/ORF) or FCGR2C^(ORF/stop) onlyexpress FcγRIIc1 (data not shown).

Distribution of FCGR2C Expression.

Next, mRNA expression of FcγRII isoforms in neutrophils, monocytes, NKcells, T cells and B cells from healthy volunteers was investigated.Transcripts for FcγRIIa, FcγRIIb2 and FcγRIIc, but not FcγRIIb1, werefound in both neutrophils and monocytes while B cells only containedFcγRIIb1 (FcγRIIb1 contains exon 6 of FCGR2B, FcγRIIb2 does not containthis exon). T cells did not express any of the FcγRII isoforms, whereas.NK cells solely expressed FcγRIIc.

Expression of FcγRIIc on NK Cells.

FCGR2A, FCGR2B and FCGR2C have 92-96% sequence homology. There are nomonoclonal antibodies available that can truly distinguish betweenFcγRIIa, FcγRIIb and FcγRIIc, rendering it difficult to quantify theprotein expression on cells. However, we found that NK cells onlyexpressed mRNA of the FcγRIIc isoform. For this reason, we examined theexpression of FcγRIIc on NK cells of individuals with theFCGR2COR^(stop/stop) genotype and the FCGR2C^(stop/stop) genotype. Itwas found that the presence of an FCGR2C-ORF allele correlates withFcγRII expression on NK cells, whereas the absence of a functionalallele corresponded with the absence of FcγRII on NK cells (FIG. 3B andTable 3). Thus, we conclude that FcγRIIc is present on NK cells.

TABLE 3 expression MLPA gene-specific PCR Sample NK cells MFI FCGR2Cstop/ORF 2B/C-386 2B-386 2B-120 2C-386 2C-120 1 no nd stop/stop W 2 nond stop/stop W 3 no nd stop/stop W 4 no nd stop/stop W 5 no nd stop/stopW 6 no nd stop/stop W 7 no nd stop/stop W 8 no nd stop/stop W 9 no ndstop/stop W 10 no nd stop/stop W 11 yes  590 ORF/stop He GC TA GC TT 12yes 1188 ORF/stop He GC TA GC TT 13 yes 1648 ORF/stop He GC TA GC TT 14yes 2602 ORF/stop He GC TA GC TT 15 yes 2123 ORF/stop He GC TA GC TT 16yes 1109 ORF/stop He GC TA GC TT 17 yes 2380 ORF/stop He GC TA GC TT 18yes 1243 ORF/stop He GC TA GC TT 19 yes 1394 ORF/stop He CC AA GC TT 20yes 1629 ORF/stop He GC TA GC TTIn Vitro Activation of NK Cells.

The FcγRIIc expression on NK cells was measured 3 times at various timeintervals (range 2-5 months) in four individuals with theFCGR2C^(ORF/stop) genotype. Minor fluctuation was observed over time(MFI±SEM: 1707±211). Subsequent testing in NK cell cultures with the NKcell activators IL-2 or IL-15 showed the ability to modulate the FcγRexpression on NK cells. We found that both IL-2 and IL-15 upregulatedthe surface expression of FcγRIIIa on NK cells. In contrast, FcγRIIcexpression was downregulated during culture, although IL-15significantly rescued the loss of FcγRIIc expression compared to eitherIL-2 or medium (FIG. 3C).

Because surface expression of FcγRIIc on neutrophils and monocytes canat present not be monitored owing to the lack of specific antibodies,the regulation of FcγRIIc mRNA in these cells was tested afterincubating neutrophils as well as PBMCs with the inflammatory activatorsLPS, GM-CSF or TNFα. In particular, GM-CSF induced a strong upregulationof FcγRIIc mRNA in neutrophils obtained from individuals with anFCGR2C^(ORF/stop) genotype, whereas this was found to a significantlylower extent in individuals with an FCGR2C^(stop/stop) genotype (p<0.01,FIG. 3D).

Redirected Antibody-Dependent Cellular Cytotoxicity.

To assess whether FcγRIIc expression on the innate immune cells wasfunctionally active, we tested the capability of the NK cells to killantibody-coated targets. In order to selectively target either FcγRIIcor FcγRIIIa on NK cells, the rADCC test was employed, involving theFcγR-bearing murine mastocytoma P815 cell line loaded with anti-FcγRIIor anti-FcγRIII antibody.

NK cells of an FCGR2C^(ORF/stop) genotype donor killed both anti-FcγRIIand anti-FcγRIII-loaded target cells (FIG. 3E). In contrast, NK cells ofan FCGR2C^(stop/stop) genotype donor were only able to killanti-FcγRIII-loaded target cells. When the expression of FcγRIIc wasreduced by culturing NK cells in the presence of activating cytokines,the effect of FcγRIIc crosslinking in the rADCC assay showed even morepronounced killing capacity in IL-2 or IL-15-activated NK cells (FIG.3F).

In conclusion, these data show that FCGR2C-ORF predisposes to ITP. Wehave shown that FcγRIIc is expressed by phagocytes and NK cells, andthat this enhances effector functions towards antibody-coated targets.Furthermore, we have shown that under inflammatory conditions (GM-CSF orIL-15), FcγRIIc function is even further enhanced. Thus, taken together,these results show that FCGR2C is a variably expressed gene highlyrelevant for immunity, and that it can serve as prognostic anddiagnostic marker to assess susceptibility and severity of infectionsand autoimmune disease.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence.txt”, created onFeb. 23, 2009. The sequence.txt file is 11 kb in size.

1. A set of probes for multiplex ligation probe amplification (MLPA)analysis of the FcγRII/FcγRIII gene cluster, the set comprising assayprobes consisting of 130-480 nucleotides wherein the assay probes havethe sequences set forth in SEQ ID NOs: 15, 16, 18, 20, 22, 23, 27-30,32-36, 39, 41, 42, 44-49, 51 and
 52. 2. A diagnostic kit for MLPAanalysis of the FcγRII/FcγRIII gene cluster, said kit comprising theassay probes of claim
 1. 3. The diagnostic kit of claim 2 comprising atleast one container having assay probes.
 4. The diagnostic kit of claim3 further comprising an additional container having assay probescomprising SEQ ID NOs: 25, 29, 31 and 38.